Equine Infectious Anemia Virus Strains Research Articles

Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.

Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes, which was relatively conserved in all the known EIAV strains. The performance of the TG-qPCR was evaluated against that of the standard qPCR (recommended by WOAH) by testing viral RNA extracted from viral supernatants of EIAVDLV2-6 and EIAVUK3, proviral DNA from peripheral blood mononuclear cells of artificially immunized horses, and virus nucleic acid from EIAV positive serum samples. The TG-qPCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the TG-qPCR assay was 1 copy/reaction for both viral RNA and proviral DNA based on the Poisson distribution. Compared to the qPCR, the TG-qPCR has better inclusivity and can detect not only Asian EIAV strains but also almost all the representative EIAV strains from other continents. The above results show that the TG-qPCR assay could serve as an effective tool for the early diagnosis of clinical EIA disease.IMPORTANCEEquine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.

Comparative analysis of CpG islands in equine infectious anemia virus strains.

Increasing evidence suggests that DNA methylation has key roles in the replication of retroviruses, including lentiviruses, and pathogenesis of diseases. However, the precise characteristics of CpG islands are not known for many retroviruses. In this study, we compared the distribution of CpG islands among strains of equine infectious anemia virus (EIAV), a lentivirus in the family Retroviridae and a model for HIV research. We identified CpG islands in 32 full-length EIAV genomic sequences obtained from the GenBank database using MethPrimer. Only one CpG island, from 100 to 120bp, was identified in the genomes of EIAV strains DV10, DLV3-A, and DLV5-10 from China, V26 and V70 from Japan, and IRE H3, IRE F2, IRE F3, and IRE F4 from Ireland. Importantly, the CpG island was located within the Rev gene, which is required for the expression of viral cis-acting elements and the production of new virions. These results suggest that the distribution, length, and genetic properties of CpG islands differ among EIAV strains. Future research should focus on the biological significance of this CpG island within rev to improve our understanding of the precise roles of CpG islands in epigenetic regulation in the species.

Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis.

Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets host immune cells, and causes a life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a highly virulent strain in vitro The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs) and found that the divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence led to disparate mitochondrial function, morphology, and metabolism. This in turn promoted the distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34-infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in the M1-polarized proinflammatory-type transformation of macrophages and the subsequent production of a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, a decrease in the mitochondrial membrane potential and impairment of the electron transport chain led to increased levels of apoptosis and reactive oxygen species. These results correlated with viral pathogenicity loss and may help provide an understanding of the key mechanism of lentiviral attenuation.IMPORTANCE Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how the mitochondrial response changes in cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in the world. EIAVDLV34 is the parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after the mitochondrial protein expression profile is altered, EIAVDLV34-infected cells are transformed into M1-polarized-type macrophages and cause inflammatory injury and that the intrinsic apoptosis pathway is activated in EIAVDLV121-infected cells. These studies shed light on how the mitochondrial protein expression profile changes between cells infected by pathogenic lentivirus strains and cells infected by attenuated lentivirus strains to drive different cellular responses, especially from inflammation to apoptosis.

Molecular characterization of Equine Infectious Anemia Viruses using targeted sequence enrichment and next generation sequencing

Equine infectious anemia virus (EIAV) is responsible of acute disease episodes characterized by fever, anemia, thrombocytopenia and anorexia in equids. The high mutation rate in EIAV genome limited the number of full genome sequences availability. In the present study, we used the SureSelect target enrichment system with Illumina Next Generation Sequencing to characterize the proviral DNA of Equine Infectious Anemia Virus (EIAV) from asymptomatic horses. This approach allows a direct sequencing of the EIAV whole genome without cloning or amplification steps and we could obtain for the first time the complete genomic DNA sequences of French EIAV strains. We analyzed their phylogenetic relationship and genetic variability by comparison with 17 whole EIAV genome sequences from different parts of the world. The results obtained provide new insights into the molecular detection of EIAV and genetic diversity of European viral strains.

Molecular characterization of equine infectious anaemia virus strains detected in England in 2010 and 2012.

Equine infectious anaemia virus (EIAV) is a retrovirus with worldwide distribution which is notifiable to the OIE. Despite its importance to the equine industry, most information regarding its biology have been obtained using only two strains (EIAVWYO and EIAVLIA ) from the USA and China, respectively. Recently full genome sequences from Ireland, Italy and Japan have been published; however, this is still not representative of the number of EIAV outbreaks experienced globally each year. The limited availability of published sequences makes design of a universal EIAV PCR difficult, hence diagnosis is solely reliant on serology. Accordingly, it is important to further investigate the re-emerging cases in other areas of the world. Here, we provide information regarding the outbreaks of EIA in England in 2010 and 2012 including the molecular characterization of strains. Full genome was obtained for two symptomatic cases but could not be resolved for the asymptomatic cases. The two British genomes from 2010 (EIAVDEV ) and 2012 (EIAVCOR ) each represent a new phylogenetic group, each differing genetically from the other available full genome sequences by 21.1%-25.5%. That the majority of new EIAV full genome sequences to be published adds another phylogenetic group indicates that the surface of EIAV global diversity is just being scratched. These data highlight that further work is needed to fully understand EIAV genetic diversity, namely the full genome sequencing of EIAV cases from a variety of locations and time points. This would aid both the use of phylogenetics in parallel with horse tracing as the epidemiological tool of disease tracking and the design of a universally applicable molecular diagnostic method.

Equine infectious anemia prevalence in feral donkeys from Northeast Brazil

Equine infectious anemia virus (EIAV) is an important cause of morbidity and mortality throughout the world. Although the virus infects all members of the Equidae the vast majority of studies have been conducted in horses (Equus caballus) with comparatively little information available for other equid species. Brazil has one of the most abundant donkey (E. asinus) populations of any nation although the economic importance of these animals is declining as transportation becomes increasingly mechanized. As a result, considerable numbers of donkeys especially in the Northeast of the country have been released and allowed pursue an almost feral existence. Consequently, this large and growing population constitutes a significant risk as a reservoir for the maintenance and transmission of important equine infectious diseases such as glanders and equine arteritis virus in addition to EIAV. This study examines the prevalence of EIA in a semi-wild donkey population from Mossoró city, in Northeast Brazil, using AGID followed by cELISA, rgp90 ELISA and immunoblot (IB). Serum samples were collected from 367 donkeys without obvious EIA clinical signs. Subsequent testing revealed seropositive rates of 1.6% (6/367) in officially approved AGID tests, 3.3% (12/367) in cELISA and 14.4% (53/367) in the rgp90 ELISA. However, 88.7% (47/53) of the rgp90 ELISA positive samples were almost certainly false reactions because they failed to react with two or more antigens in IB. Consequently, the rpg90 ELISA has a similar sensitivity to AGID with donkey serum samples. Such high false positive rates have not been observed previously with serum samples from horses. Another highly significant finding is that 56.9% (33/58) of the donkey serum samples tested in IB had reactivity to EIAV p26 only. Although this could result from recent infection with the virus, it has been found that in some equids p26 only reactivity persists for extensive periods of time suggesting exposure to antigens possessing cross-reactive determinants or EIAV strains with envelope glycoproteins that are different from any that have been previously characterized and so undetectable by current IB techniques.

A pilot study on interaction between donkey tetherin and EIAV stains with different virulent and replication characteristics.

Tetherin (BST-2) is an important host restriction factor that can inhibit the release of a diverse array of enveloped viruses from infected cells. Conversely, to facilitate their release and spread, many viruses have evolved various strategies to overcome the antiviral effect of tetherin in a species-specific manner. During the development of an attenuated equine infectious anemia virus (EIAV) vaccine in our laboratory, we found that serial passage of a field-isolated virulent EIAV strains in horse and donkey as well as the cultivated donkey cells, produces several typical EIAV strains, including EIAVDV, EIAVDLV, and EIAVFDDV, which exhibit distinct virulence and replication features invivo and invitro. However, the role of host restriction factors in EIAV evolution during the serial passage is not well understood. This study aimed to evaluate whether these newly generated strains adapt differently to donkey tetherin (do-tetherin) based on their virulence. We found that do-tetherin exerts an inhibition on the release of the viral particles produced by all three strains, albeit with varying intensity: EIAVDV<EIAVDLV<EIAVFDDV. Additionally, all three EIAV strains could counteract the restriction mediated by do-tetherin via their envelope proteins (Env) with varying strength: EIAVDV>EIAVDLV>EIAVFDDV. These results indicate that donkey tetherin is involved in shaping of EIAV evolution during serial passage.

Identification and characterization of a common B-cell epitope on EIAV capsid proteins.

The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies(MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this study, we characterized two monoclonal antibodies (9H8 and 1G11 MAbs) against EIAV p26. Two B-cell epitopes are located in amino acid residues, 73NLDKIAEE81 (HE) and 199KNAMRHLRPEDTLEEKMYAC218 (GE) for the 9H8 and 1G11 MAbs, respectively. The 1G11 epitope (GE) varied among viruses isolated worldwide but can be recognized by anti-EIAV sera from different regions, including China, the USA, and Argentina. Meanwhile, 1G11 MAb could react with the mutants of almost all the EIAV strains. Furthermore, we found that the histidine at position 204 (H204), leucine at position 205 (L205), and aspartic acid at position 209 (D209) of EIAV p26 individually played pivotal roles in binding with the 1G11 MAb. Our results revealed that the GE peptide might be a common B-cell binding epitope of EIAV antibodies. This is also the first report to identify a broad-spectrum monoclonal antibody (1G11) against p26 of EIAV. These findings may provide a useful basis for the development of new diagnostic assays for EIAV.

Insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45.

An effective vaccine against acquired immune deficiency syndrome is still unavailable after dozens of years of striving. The glycoprotein gp41 of human immunodeficiency virus is a good candidate as potential immunogen because of its conservation and relatively low glycosylation. As a reference of human immunodeficiency virus gp41, gp45 from equine infectious anemia virus (EIAV) could be used for comparison because both wild-type and vaccine strain of EIAV have been extensively studied. From structural studies of these proteins, the conformational changes during viral invasion could be unveiled, and a more effective acquired immune deficiency syndrome vaccine immunogen might be designed based on this information.

High genetic diversity of equine infectious anaemia virus strains from Slovenia revealed upon phylogenetic analysis of the p15 gag gene region.

The equine infectious anaemia virus (EIAV), which belongs to the Retroviridae family, infects equids almost worldwide. Every year, sporadic EIAV cases are detected in Slovenia. To characterise the Slovenian EIAV strains in the p15 gag gene region phylogenetically in order to compare the Slovenian EIAV strains with EIAV strains from abroad, especially with the recently published European strains. Cross-sectional study using material derived from post mortem examination. In total, 29 EIAV serologically positive horses from 18 different farms were examined in this study. Primers were designed to amplify the p15 gag gene region. Amplicons of 28 PCRs were subjected to direct DNA sequencing and phylogenetic analysis. Altogether, 28 EIAV sequences were obtained from 17 different farms and were distributed between 4 separate monophyletic groups and 9 branches upon phylogenetic analysis. Among EIAV strains from abroad, the closest relatives to Slovenian EIAV strains were European EIAV strains from Italy. Phylogenetic analysis also showed that some animals from distantly located farms were most probably infected with the same EIAV strains, as well as animals from the same farm and animals from farms located in the same geographical region. This is the first report of such high genetic diversity of EIAV strains from one country. This led to speculation that there is a potential virus reservoir among the populations of riding horses, horses kept for pleasure and horses for meat production, with some farmers or horse-owners not following legislation, thus enabling the spread of infection with EIAV. The low sensitivity of the agar gel immunodiffusion test may also contribute to the spread of infection with EIAV, because some infected horses might have escaped detection. The results of the phylogenetic analysis also provide additional knowledge about the highly heterogeneous nature of the EIAV genome.

A unique evolution of the s2 gene of equine infectious anemia virus in hosts correlated with particular infection statuses.

Equine infectious anemia virus (EIAV) is a member of the Lentivirus genus in the Retroviridae family that exhibits a genomic structure similar to that of HIV-1. The S2 accessory proteins play important roles in viral replication in vivo and in viral pathogenicity; however, studies on S2 evolution in vivo are limited. This study analyzed the evolutionary characteristics of the S2 gene of a pathogenic EIAV strain, EIAVLN40, in four experimentally infected horses. The results demonstrated that 14.7% (10 of 68 residues) of the stable amino acid mutations occurred longitudinally in S2 during a 150-day infection period. Further analysis revealed that six of the ten mutated residues were positively selected during the infection. Alignment and phylogenetic analyses showed that the S2 gene sequences of viruses isolated from the infected horses at the early stage of EIAVLN40 infection were highly homologous and similar to the vaccine-specific sequence. The S2 gene variants isolated from the febrile episodes and late phase of infection became homologous to the S2 gene sequence of the inoculating EIAVLN40 strain. Our results indicate that the S2 gene evolves in diversity and divergence in vivo in different stages of EIAV infection and that this evolution correlates with the pathogenicity of the virus.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNβ expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0082-y) contains supplementary material, which is available to authorized users.

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain

Comprehensive analysis of the overall codon usage patterns in equine infectious anemia virus

BackgroundEquine infectious anemia virus (EIAV) is an important animal model for understanding the relationship between viral persistence and the host immune response during lentiviral infections. Comparison and analysis of the codon usage model between EIAV and its hosts is important for the comprehension of viral evolution. In our study, the codon usage pattern of EIAV was analyzed from the available 29 full-length EIAV genomes through multivariate statistical methods.FindingEffective number of codons (ENC) suggests that the codon usage among EIAV strains is slightly biased. The ENC-plot analysis demonstrates that mutation pressure plays a substantial role in the codon usage pattern of EIAV, whereas other factors such as geographic distribution and host translation selection also take part in the process of EIAV evolution. Comparative analysis of codon adaptation index (CAI) values among EIAV and its hosts suggests that EIAV utilize the translational resources of horse more efficiently than that of donkey.ConclusionThe codon usage bias in EIAV is slight and mutation pressure is the main factor that affects codon usage variation in EIAV. These results suggest that EIAV genomic biases are the result of the co-evolution of genome composition and the ability to evade the host’s immune response.

Understanding virus–host dynamics following EIAV infection in SCID horses

We develop a mathematical model for the interaction between two competing equine infectious anemia virus strains and neutralizing antibodies. We predict that elimination of one or both virus strains depends on the initial antibody levels, the strength of antibody mediated neutralization, and the persistence of antibody over time. We further show that the ability of a subdominant, neutralization resistant virus to dominate the infection transiently or permanently is dependent on the antibody-mediated neutralization effect. Finally, we determine conditions for persistence of both virus strains. We fit our models to virus titers from horses (foals) with severe combined immunodeficiency to estimate virus–host parameters and to validate analytical results.

Reverse mutation of the virulence-associated S2 gene does not cause an attenuated equine infectious anemia virus strain to revert to pathogenicity

The contribution of S2 accessory gene of equine infectious anemia virus (EIAV) to the virulence of pathogenic strains was investigated in the present study by reverse mutation of all four consensus S2 mutation sites in an attenuated EIAV proviral strain, FDDV3-8, to the corresponding sequences of a highly pathogenic strain DV117. The S2 reverse-mutated recombinant strain FDDVS2r1-2-3-4 replicated with similar kinetics to FDDV3-8 in cultivated target cells. In contrast to the results of other studies of EIAV with dysfunctional S2, reverse mutation of S2 only transiently and moderately increased the plasma viral load of inoculated horses, and induction of transient immunosuppression did not boost viral pathogenicity. In addition, inoculation of FDDVS2r1-2-3-4 induced partial protection to a challenge pathogenic virus. These results suggest that the attenuated EIAV vaccine strain with multiple mutations in multiple genes will not easily revert to a virulent phenotype.

Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia?

To improve the efficiency of the National equine infectious anaemia (EIA) surveillance program in Italy, a three-tiered diagnostic system has been adopted. This procedure involves initial screening by ELISA (Tier 1) with test-positive samples confirmed by the agar gel immunodiffusion test (AGIDT) (Tier 2) and, in the case of ELISA positive/AGIDT negative results, final determination by immunoblot (IB) (Tier 3). During this evaluation, 74,880 samples, principally collected from two Regions of Central Italy (Latium and Abruzzo) were examined, with 44 identified as negative in AGIDT but positive in both ELISA and IB. As the majority of these reactions occurred in mules, an observational study was conducted in this hybrid equid species to investigate if there is a correlation between plasma-associated viral loads and serological reactivity, to test the hypothesis that false-negative or very weak positive AGIDT results are associated with elite control of EIA virus (EIAV) replication accompanied by reduced transmission risks. The study animals consisted of 5 mules with positive AGIDT readings, along with another 5 giving negative or very weak positive results in this test. All mules were seropositive in Elisa and IB. Samples were collected routinely during an initial 56-day observation period, prior to dexamethasone treatment lasting 10 days, to determine the effect of immune suppression (IS) on clinical, humoral and virological responses. All mules were monitored for a further 28 days from day 0 of IS. None of the animals experienced relevant clinical responses before IS and there were no significant changes in antibody levels in ELISA, IB or AGIDT. However, plasma-associated viral-RNA (vRNA) loads, as determined using TaqMan(®) based RT-PCR, showed unexpectedly high sample to sample variation in all mules, demonstrating host-mediated control of viral replication is not constant over time. Furthermore, there was no apparent correlation between vRNA loads and antibody reactivity in serological tests. Analysis of PCR products established all mules were infected with viruses possessing nucleotide sequence similarity, varying from 77 to 96%, to previously identified European EIAV strains. Following IS, all mules showed increases in plasma-associated vRNA loads, suggesting control of EIAV replication is mediated by immune responses in this hybrid species. However, only three mules showed anamnestic humoral responses to rises in viral loads, as defined by at least a four-fold increase in ELISA titre, while two remained AGIDT-negative. This study demonstrates that viral loads in equids with consistent ELISA/IB positive-AGIDT negative to very weak positive test results (Group N) can be equivalent to those that produce clearly positive results in all three serologic tests (Group P). Therefore, such animals do not pose inherently lower risks for the transmission of EIAV. Consequently, the exclusive use of the AGIDT, as prescribed by the World Organization of Animal Health (OIE) for diagnosis of EIA prior to the international movement of horses, can report as negative some EIAV-infected equids. These results dramatically underscore the necessity of combining the specificity of AGIDT with tests with higher sensitivity, such as the ELISA and the power of the IB to enhance the accuracy of EIA diagnosis.

Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates

In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward primers and one reverse primer in the polymerase chain reaction enabled the amplification of all EIAV strains used in this study. However, further study is required to confirm these primers as universal for all EIAV strains. The nucleotide sequence of gag is considered highly conserved, as evidenced by the use of gag-encoded capsid proteins as a common antigen for the detection of EIAV in serological tests. However, significant sequence variation in the gag genes of different EIAV strains was found in the current study.

Epidemiology and Genetic Characterization of Equine Infectious Anaemia Virus Strains Isolated in Belgium in 2010

In January 2010, the United Kingdom notified cases of equine infectious anaemia (EIA) in two horses introduced from Belgium. The animals came from one assembly centre in Romania and had transited through Belgium with 16 other horses. Nine of them, bought by a Belgian horse breeder, were investigated in Belgium and revealed one additional EIA-positive animal. Afterwards, the Belgian Federal Agency for the Safety of the Food Chain (FASFC) organized a serological EIA survey of the horses introduced into Belgium from Romania between 2007 and 2009. Among the 95 horses identified, six additional serological positive cases were found that had been introduced into Belgium in 2008 (n=4) and in 2009 (n=2). The survey was extended to the horses in contact with the positive cases, but all contact animals were negative, indicating the absence of transmission. Virological examination performed on tissue samples collected from two seropositive animals demonstrated the presence of viral DNA of EIA virus. Phylogenetic analysis based on the sequences of EIA virus gag gene clustered the Belgian isolates with Romanian strains isolated in 2009. The presumption of a common Belgian origin could be rejected.